CN111662869A - Immunomagnetic bead for separating red blood cells and preparation method and application thereof - Google Patents
Immunomagnetic bead for separating red blood cells and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an immunomagnetic bead for separating red blood cells, a preparation method and application thereof, and relates to the technical field of immunomagnetic bead preparation. The preparation method of the immunomagnetic beads comprises the following steps: s1, activating magnetic beads by using an activating agent, wherein the weight ratio of the magnetic beads to the activating agent is 1: 0.1-5; the activation time is 5-30 min; step S2, adding an anti-erythrocyte antibody into the activated product obtained in the step S1, and carrying out ultrasonic treatment; and S3, adding a sealing agent into the product obtained in the step S2 for sealing, and performing strong magnetic separation to obtain the immunomagnetic beads for separating the red blood cells. The immunomagnetic beads obtained by the method are used for separating red blood cells in blood plasma, can complete the separation of the blood cells within 1-2min, save time, provide a foundation for rapid detection, and simultaneously avoid hemolysis caused by factors such as centrifugation and the like, thereby improving the accuracy of detection results.
Description
Technical Field
The invention belongs to the technical field of immunomagnetic bead preparation, and particularly relates to an immunomagnetic bead for separating red blood cells, and a preparation method and application thereof.
Background
The immune magnetic bead sorting (IMS) is a method for sorting cells that has recently been developed, in which immunoreactive antibodies are coated on the surfaces of magnetic beads to perform antigen-antibody reaction, thereby forming "antigen-antibody-magnetic bead" immune complexes on the surfaces of cells. Once placed under a strong magnetic field, these cells bound to the magnetic beads move in a targeted manner, grouping the immune complexes with other unbound cells. When the magnetic beads with superparamagnetism are separated from the magnetic field, the magnetism disappears immediately, thereby achieving the purpose of selecting specific cells positively or negatively.
In clinical examination, blood components are examined by separating blood serum or plasma from blood cell-containing components in whole blood. Clinically, the separation of plasma is usually performed by separating red blood cells, white blood cells, and platelets from plasma by centrifugation or standing. However, the red blood cells interfere with the capture of antigen by the immunomagnetic beads in the conventional kit, and the rupture of the red blood cells causes measurement errors caused by a large amount of hemoglobin, high concentration of ions or enzyme release, thereby reducing the accuracy of sample detection.
In the traditional separation method, the standing separation takes too long; when the centrifugation operation is improper, the blood cells may be broken, which is not favorable for rapid detection. Meanwhile, the half-life of the molecular marker of some projects is short, and the content of the molecules to be detected in the blood plasma can be reflected more truly and accurately by quickly separating blood cells for detection; in patients with myocardial infarction, a rapid and accurate result is more beneficial in striving for valuable time for the patient to have an effective treatment within the first minute.
Disclosure of Invention
The invention mainly aims to provide an immunomagnetic bead for separating red blood cells, and a preparation method and application thereof, and aims to solve the technical problems that the conventional separation method has the disadvantages of long standing separation time, rupture of blood cells caused by improper centrifugal operation, and inconvenience for rapid detection.
The purpose of the invention and the technical problem to be solved are realized by adopting the following technical scheme. The invention provides a preparation method of immunomagnetic beads for separating red blood cells, which comprises the following steps:
s1, activating magnetic beads by using an activating agent, wherein the weight ratio of the magnetic beads to the activating agent is 1: 0.1-5; the activation time is 5-30 min;
step S2, adding an anti-erythrocyte antibody into the activated product obtained in the step S1, and carrying out ultrasonic treatment;
and S3, adding a sealing agent into the product obtained in the step S2 for sealing, and performing strong magnetic separation to obtain the immunomagnetic beads for separating the red blood cells.
The object of the present invention and the technical problems solved thereby can be further achieved by the following technical measures.
Preferably, in the preparation method of immunomagnetic beads for separating erythrocytes, the magnetic beads are microspheres coated with ferroferric oxide or ferric oxide, and the surfaces of the microspheres have carboxyl, amino or hydroxyl groups.
Preferably, in the preparation method of immunomagnetic beads for separating erythrocytes, the magnetic beads are polystyrene microspheres coated with ferroferric oxide.
Preferably, in the method for preparing immunomagnetic beads for separating red blood cells, the particle size of the magnetic beads is 1-5 μm.
Preferably, the method for preparing immunomagnetic beads for separating red blood cells comprises the step of preparing an activator, wherein the activator comprises at least one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, glutaraldehyde, periodate, and chloramine-T.
Preferably, in the method for preparing immunomagnetic beads for separating erythrocytes, in step S2, the mass ratio of the anti-erythrocyte antibodies to the magnetic beads is: 1: 10-1000.
Preferably, in the preparation method of immunomagnetic beads for separating red blood cells, in step S3, the blocking agent is at least one selected from the group consisting of 1-5 wt% bovine serum albumin solution, 0.1-5 wt% casein solution and 10-50mM glycine.
Preferably, in the preparation method of immunomagnetic beads for separating red blood cells, the step S3 further includes: washing the obtained immunomagnetic beads for separating the red blood cells, adding the immunomagnetic beads into a preservation solution, uniformly dispersing, dividing into small parts according to the required amount, and carrying out vacuum drying, wherein the preservation solution comprises 50mM of tris (hydroxymethyl) aminomethane and 0.5 wt% of bovine serum albumin, and the pH value of the preservation solution is 7.8.
Preferably, the method for preparing immunomagnetic beads for separating red blood cells comprises the following steps: and carrying out vacuum freeze-drying treatment on the immunomagnetic beads under the pressure of 10-30Pa and the temperature of below-30 ℃.
The object of the present invention and the technical problem to be solved are also achieved by the following technical means. The invention provides an immunomagnetic bead for separating red blood cells, which is prepared by the preparation method of any one of the preceding methods.
The object of the present invention and the technical problem to be solved are also achieved by the following technical means. The invention provides an application of the immunomagnetic beads in separation of red blood cells in blood plasma.
By the technical scheme, the immunomagnetic beads for separating the red blood cells and the preparation method and the application thereof provided by the invention at least have the following advantages:
the method of the invention enables the magnetic beads and the anti-erythrocyte antibodies to be better combined by activating the magnetic beads, removes the uncombined substances after being sealed by the sealing agent, and obtains the immunomagnetic beads for separating erythrocytes through centrifugal separation. The immunomagnetic beads obtained by the method are used for separating red blood cells in blood plasma, can complete the separation of the blood cells within 1-2min, save time, provide a foundation for rapid detection, and simultaneously avoid hemolysis caused by factors such as centrifugation and the like, thereby improving the accuracy of detection results.
The obtained immunomagnetic beads can be connected with red blood cells, blood plasma and blood cells can be simply and quickly separated through magnetic separation, and the blood cells are separated through immunoreaction, so that the immunomagnetic beads have no influence on substances to be detected. The immunomagnetic beads are used for separating and removing the red blood cells, the operation is simple, the time for separating the red blood cells can be greatly shortened, the occurrence of hemolysis is greatly reduced, and the immunomagnetic beads can be applied to full-automatic and semi-automatic chemiluminescence apparatuses and matched with corresponding in-vitro diagnostic kits for detecting whole blood samples.
The immunomagnetic beads have long shelf life, can always keep higher activity, are simple and portable, and save space.
The method is simple to operate and easy to realize, and the obtained immunomagnetic beads can be combined with a POCT luminescence kit and used for whole blood detection.
The immunomagnetic beads can help a chemiluminescence detection device or an immunoturbidimetric detection device to carry out whole blood detection, and are more convenient and wider in application range while ensuring detection accuracy; the immunomagnetic beads are added, so that the influence of the blood cells in the whole blood during detection can be effectively reduced, and the detection result is more accurate.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is a schematic diagram of a whole blood sample before being added with immunomagnetic beads according to an embodiment of the present invention;
FIG. 2 shows a schematic diagram of a whole blood sample after adding immunomagnetic beads according to an embodiment of the present invention.
Detailed Description
To further illustrate the technical means and effects of the present invention for achieving the predetermined objects, the following detailed description will be made with reference to the accompanying drawings and preferred embodiments of the immunomagnetic beads for separating red blood cells, the preparation method thereof, and the application thereof. In the following description, different "one embodiment" or "an embodiment" refers to not necessarily the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
One embodiment of the present invention provides a method for preparing an immunomagnetic bead for separating red blood cells, which comprises:
s1, cleaning magnetic beads, removing impurities, adding a phosphate buffer solution into the cleaned magnetic bead precipitate to enable the pH value to be within the range of 5.0-6.5, preferably pH 6.0, and the acidic condition is more favorable for activating groups, ultrasonically mixing the magnetic bead solution uniformly, wherein the purpose of ultrasonic is to mix uniformly, preferably the ultrasonic condition is 40kHz and 6min, adding an activating agent into the magnetic beads, ultrasonically mixing uniformly, preferably the ultrasonic condition is 40kHz and 6min, the weight ratio of the magnetic beads to the activating agent is 1:0.1-5, then placing the activated solution on a shaking bed, and carrying out oscillation reaction at 37 ℃ and 100rpm for 24 min.
Step S2, adding an anti-erythrocyte antibody into the activated product obtained in the step S1, and carrying out ultrasonic treatment; the ultrasonic treatment enables the magnetic beads to react with the anti-erythrocyte antibodies more uniformly and be combined more fully, and the microbeads are prevented from sinking and aggregating.
And S3, adding a sealing agent into the product obtained in the step S2 for sealing, and performing strong magnetic separation to obtain the immunomagnetic beads for separating the red blood cells.
The magnetic beads in the embodiment of the present invention are carrier microspheres having a certain magnetism and a special structure and a volume of several nanometers to several tens of micrometers, which are formed by combining magnetic inorganic ions and organic polymers by a certain method. The carrier microsphere is usually iron oxide or iron sulfide, a layer of high polymer material is wrapped outside the core, and the outermost layer is a functional group, so that the carrier microsphere has different physical properties of hydrophobicity, hydrophilicity, nonpolar, polarity, positive charge, negative charge and the like; meanwhile, the magnetic response is realized, and the magnetic steering type magnetic sensor has magnetic steering under the action of an external magnetic field. In order to meet the requirement of subsequent magnetic separation, the magnetic bead provided by the invention has the following characteristics: the particle size is small, the specific surface area is large, and the adsorption capacity is large; the physical and chemical properties are stable, the mechanical strength is high, and the service life is long; having an activatable reactive group for the immobilization of an affinity ligand; the particle size is uniform, and a monodisperse system can be formed; good suspension property and convenient effective reaction.
After the magnetic beads are activated by the activator, active groups such as carboxyl or hydroxyl on the surfaces of the magnetic beads are opened, so that the magnetic beads and the antibodies can be combined more quickly, and the reactivity of the magnetic beads is increased.
In some embodiments, in said step S2, the time of said activating is 5-30 min; the activator comprises at least one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, glutaraldehyde, periodate, and chloramine-T.
In some embodiments, the magnetic beads are microspheres coated with ferroferric oxide or ferric oxide, and the surfaces of the microspheres have active groups such as carboxyl, amino or hydroxyl.
In this embodiment, the magnetic beads are organic polymers coated with magnetic inorganic ions, and the surface of the magnetic beads is microspheres with active groups, such as amino ferric oxide magnetic microspheres, and ferroferric oxide magnetic microspheres with carboxyl groups.
In some embodiments, the magnetic beads are polystyrene microspheres wrapped with ferroferric oxide.
The magnetic beads can be quickly combined with anti-erythrocyte antibodies after being activated and generate coagulation under the action of magnetic force.
In some embodiments, the magnetic beads have a particle size of 1 to 5 μm.
The magnetic beads with the particle size of more than 5 μm are not easy to couple with the antibody, the magnetic beads with the particle size of less than 1 μm are not easy to separate by strong magnetism, and the preferred particle size of the magnetic beads is 2-4 μm.
In some embodiments, the mass ratio or volume ratio of the anti-erythrocyte antibody to the activated magnetic bead is: 1: (10-1000).
The principle of combining anti-erythrocyte antibodies with magnetic beads: the magnetic beads contain carboxyl groups, and the reactivity of the magnetic beads can be increased after activation, so that the magnetic beads can react with amino groups in the anti-erythrocyte antibodies.
In some embodiments, in step S3, the blocking agent is selected from at least one of a 1-5 wt% bovine serum albumin solution, a 0.1-5 wt% casein solution, and 10-50mM glycine. The sealing time is 5min-30 min; the rotation speed is 100-200 rpm.
Further, the preparation method of the above embodiment further includes:
and cleaning the obtained immunomagnetic beads for separating the red blood cells, adding the immunomagnetic beads into a preservation solution, uniformly dispersing, dividing into small parts according to the required amount, and performing vacuum drying.
The cleaning agent used in the step is the same as the sealing agent in the step S3, the cleaning agent is added, the magnetic beads are subjected to ultrasonic resuspension, then strong magnetic separation is carried out, and the cleaning is carried out twice. And (4) subpackaging the obtained product into penicillin bottles according to the ratio of the preservation solution to the magnetic beads, and freeze-drying.
The preservation solution is typically Tris solution containing BSA, and the immunomagnetic beads added with the preservation solution can be preserved for 14 days at 4 ℃.
In some embodiments, the preservation solution is 50mM tris and 0.5 wt% bovine serum albumin, and the pH of the preservation solution is 7.8.
In some embodiments, the vacuum drying comprises: and carrying out vacuum freeze-drying treatment on the immunomagnetic beads under the pressure of 10-30Pa and the temperature of below-30 ℃.
The embodiment of the invention adopts vacuum freeze drying, so that the original characteristics of the immunomagnetic beads can be better maintained, and the loss of quality is reduced. The vacuum drying is carried out to evaporate and boil simultaneously, the vaporization speed is accelerated, the vacuum pumping is carried out to quickly pump vaporized steam, a negative pressure state is formed around the magnetic beads, the surfaces of the magnetic beads and surrounding media form a large moderate gradient, the vaporization is accelerated, and the purpose of quick drying is achieved.
The method of the invention enables the magnetic beads and the anti-erythrocyte antibodies to be better combined by activating the magnetic beads, removes the uncombined substances after being sealed by the sealing agent, and obtains the immunomagnetic beads for separating erythrocytes through centrifugal separation. The method of the invention is simple to operate and easy to realize.
The invention also provides an immunomagnetic bead for separating red blood cells, wherein the immunomagnetic bead is prepared by the preparation method.
The obtained immunomagnetic beads can be combined with a POCT luminescence kit and used for whole blood detection. The sample used in the present invention is fresh whole blood, and can be used alone as a whole blood processing apparatus or as a part of a POCT chemiluminescent reagent.
One embodiment of the invention also provides application of the immunomagnetic beads in separation of red blood cells in plasma.
Referring to fig. 1-2, which show the application of immunomagnetic beads in separating red blood cells from plasma according to an embodiment of the present invention, fig. 1 is a schematic diagram before adding the immunomagnetic beads, a whole blood sample to be measured (including plasma 2 and red blood cells 3) is placed in a glass tube 1, the obtained immunomagnetic beads 4 are added, and 0.1-10mg of immunomagnetic beads are added to 1mL of the whole blood sample; fig. 2 is a schematic diagram showing the process after the immunomagnetic beads are added, after the immunomagnetic beads 4 are added, the erythrocytes 3 in the glass tube 1 are combined with the immunomagnetic beads 4 to form erythrocyte-magnetic bead immunocomplexes 5, the bottom of the glass tube 1 is placed on a strong magnetic magnet 6, under the action of the strong magnetic magnet, the erythrocyte-magnetic bead immunocomplexes 5 in the glass tube 1 move to the bottom of the glass tube 1, and the plasma 2 remains on the upper layer, so that the plasma 2 and the erythrocytes 3 are separated to obtain the plasma 2 for detection.
The working principle of the invention is as follows: the anti-erythrocyte antibody is connected with the magnetic beads through a chemical means to obtain immunomagnetic beads, when the immunomagnetic beads are contacted with whole blood, the immunomagnetic beads react with the erythrocyte to generate erythrocyte-anti-erythrocyte antibody-magnetic bead compounds, and then the aim of removing the erythrocyte is achieved through magnetic separation. The invention is based on the immunomagnetic bead separation technology, and can simply and rapidly separate blood plasma and blood cells.
The immunomagnetic bead separation technology is a specific magnetic separation technology. The immunomagnetic beads can be combined with active proteins (antibodies) and can also be attracted by magnets, after certain treatment, the antibodies can be combined on the magnetic beads to become carriers of the antibodies, after the antibodies on the magnetic beads are combined with specific antigen substances, antigen-antibody-magnetic bead immune complexes are formed, and the complexes are mechanically moved under the action of magnetic force to separate the complexes from other substances, so that the purpose of separating specific antigens is achieved. The invention relates to an immunomagnetic bead action mode which is a direct method, wherein firstly, an antibody is used for coating a magnetic bead, so that the antibody is combined with the magnetic bead (physical adsorption or chemical combination), then an antigen substance is added, the antibody and the magnetic bead are combined to form a compound, and the compound is separated from other substances under the action of magnetic force.
The invention can provide assistance for whole blood testing with partial testing methodology. If the sample is detected by using equipment related to a chemiluminescence method and an immunoturbidimetry method, and if the sample type is whole blood, blood cells in the sample have a large influence on a detection result, when the detection is generally performed by using methods such as chemiluminescence or immunoturbidimetry, the sample needs to be blood plasma or blood serum, and the whole blood cannot be detected; the immunomagnetic beads are added, so that the influence of the blood cells in the whole blood during detection can be effectively reduced, and the detection result is more accurate.
The present invention will be further described with reference to the following specific examples, which should not be construed as limiting the scope of the invention, but rather as providing those skilled in the art with certain insubstantial modifications and adaptations of the invention based on the teachings of the invention set forth herein.
The following materials or reagents, unless otherwise specified, were all purchased.
Example 1
A preparation method of immunomagnetic beads for separating red blood cells specifically comprises the following steps:
1.1.1 washing of magnetic beads:
the original concentration of the magnetic beads was 2.0 wt%, 10mM phosphate buffer (pH 6.0) was added, the mixture was subjected to strong magnetic centrifugation, the supernatant was discarded, and the process was repeated twice to obtain a magnetic bead precipitate.
1.1.2 activation:
1) preparing an activating agent solution:
the activator selects N-hydroxysuccinimide and 1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide with the weight ratio of 1:1, and 10mM (pH 6.0) phosphate buffer is respectively used for preparing 500mg/mL solution for standby;
2) activation of magnetic beads:
adding 10mM phosphate buffer (pH 6.0) into the washed magnetic bead precipitate, ultrasonically mixing (40kHz, 6min) to obtain a 100mg/mL magnetic bead solution, mixing the 500mg/mL N-hydroxysuccinimide prepared in the previous step 1) with the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide mixed solution (magnetic beads, adding N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide in a weight ratio of 1:2.5:2.5) and ultrasonically mixing uniformly (40kHz, 6min), wherein the concentration of magnetic beads in the obtained solution is 10mg/mL, the concentration of N-hydroxysuccinimide is 25mg/mL, and the concentration of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is 25 mg/mL; the solution was then placed on a shaker and reacted at 37 ℃ for 24min with shaking at 100 rpm.
3) Activated magnetic bead centrifugation:
the magnetic bead solution after completion of activation was subjected to strong magnetic centrifugation using 10mM phosphate buffer (pH 6.0), and the supernatant was discarded and repeated twice.
1.1.3 coupling:
adding 10mM phosphate buffer (pH 6.0) into the magnetic bead precipitate obtained by activation centrifugation, and ultrasonically mixing (40kHz, 6min) to make the final concentration of the magnetic bead be 10 mg/mL; then adding an anti-erythrocyte antibody, wherein the weight ratio of the magnetic beads to the anti-erythrocyte antibody is 100:1, ultrasonically mixing uniformly (40kHz, 6min), placing on a shaking table, and carrying out oscillation reaction for 4h at 37 ℃ and 100 rpm. In the reaction process, the mixture is ultrasonically mixed once every half hour (40kHz, 6min), so that the generation of precipitates is avoided.
1.1.4 blocking:
after the labeling is finished, 50mM glycine solution (pH 7.4) with the same volume as the solution is added into the magnetic bead solution, the mixture is ultrasonically mixed (40kHz, 6min), the mixture is placed on a shaker, and the shaking reaction is carried out at 37 ℃ and 100rpm for 14 min. After the reaction was completed, the reaction was centrifuged and washed with 50mM glycine solution (pH 7.4) at 14000rpm for 9min, and the supernatant was discarded and repeated twice.
1.1.5 preservation:
to the magnetic bead pellet obtained after the completion of the blocking centrifugation, a stock solution (50mM Tris, 0.5 wt% BSA, pH7.8) was added so that the final magnetic bead concentration became 5mg/ml, and the mixture was sonicated (40kHz, 6min) and stored at 4 ℃.
1.1.6 vacuum drying:
and ultrasonically mixing the magnetic bead storage solution uniformly (40kHz, 6min), subpackaging the magnetic bead storage solution into 5mL penicillin bottles according to 0.2 mL/bottle, sealing the vials by using gauze, putting the vials into a vacuum freeze dryer, and carrying out vacuum freeze drying for 12h at 10Pa and-50 ℃. The reagent can be stored at 4 deg.C for two years.
Example 2
When the immunomagnetic beads obtained in the embodiment 1 are used for separating red blood cells in blood plasma, 0.5mL of whole blood is added into the penicillin bottle in the embodiment 4, the mixture is shaken up and then placed on a magnetic frame or a magnet, and the upper layer of blood plasma is obtained after 2 min.
Example 3
Comparing the magnetic bead separation method with the standing method, and detecting the result difference by using a PCT kit.
Respectively taking 20 parts of fresh anticoagulated whole blood, uniformly dividing the anticoagulated whole blood into two groups, adding the immunomagnetic beads obtained in the embodiment 1 into one group, and then placing the groups on a magnetic frame to be marked as an immunomagnetic bead group; the other group is naturally placed with separated plasma and is marked as a natural placing group. After 2min, basically settling blood cells of the immunomagnetic bead group; while the natural setting group basically takes 20-30min to get plasma.
The plasma obtained from the two groups is simultaneously detected by using a PCT kit, and the detection results of the two groups are compared without obvious difference and are listed in Table 1.
TABLE 1
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Magnetic bead group (ng/ml) | 0.21 | 0.07 | 0.14 | 0.55 | 0.29 | 1.07 | 0.88 | 0.10 | 0.32 | 0.64 |
| Placing group (ng/ml) | 0.20 | 0.07 | 0.15 | 0.57 | 0.30 | 1.09 | 0.86 | 0.11 | 0.30 | 0.66 |
As can be seen from Table 1, the method for magnetically separating blood cells by using the immunomagnetic beads obtained by the invention is simple and effective, can rapidly separate blood cells to obtain blood plasma, and has no residue and no influence on substances to be detected.
Example 4
A preparation method of immunomagnetic beads for separating red blood cells specifically comprises the following steps:
4.1.1 magnetic bead washing:
the original concentration of the magnetic beads was 5.0 wt%, and the magnetic beads were precipitated by strongly magnetic centrifugation using 10mM phosphate buffer (pH7.0), discarding the supernatant, and repeating the process twice.
4.1.2 activation:
1) preparing an activating agent solution:
the activating agent is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, and the solution with the concentration of 250mg/mL is prepared by respectively using 10mM (pH7.0) phosphate buffer solution for later use;
2) magnetic bead activation:
adding 10mM phosphate buffer (pH7.0) into the washed magnetic bead precipitate, ultrasonically mixing the mixture uniformly (40kHz, 6min) to obtain a 100mg/mL magnetic bead solution, adding 250mg/mL of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide solution (the weight ratio of the magnetic beads to the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is 1:5) prepared in the previous step 1), ultrasonically mixing the mixture uniformly (40kHz, 6min), wherein the concentration of the magnetic beads in the obtained solution is 10mg/mL, and the concentration of the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is 50 mg/mL; the solution was then placed on a shaker and reacted at 37 ℃ for 24min with shaking at 100 rpm.
3) Activated magnetic bead centrifugation:
the magnetic bead solution after completion of activation was subjected to strong magnetic centrifugation using 10mM phosphate buffer (pH7.0), and the supernatant was discarded and repeated twice.
4.1.3 coupling:
adding 10mM phosphate buffer (pH7.0) into the magnetic bead precipitate obtained by activation centrifugation, and ultrasonically mixing (40kHz, 6min) to make the final concentration of the magnetic bead be 10 mg/mL; then adding an anti-erythrocyte antibody, wherein the weight ratio of the magnetic beads to the anti-erythrocyte antibody is 200:1, ultrasonically mixing uniformly (40kHz, 6min), placing on a shaking table, and carrying out oscillation reaction for 2h at 37 ℃ and 100 rpm. In the reaction process, the mixture is ultrasonically mixed once every half hour (40kHz, 6min), so that the generation of precipitates is avoided.
4.1.4 blocking:
after the labeling is finished, a 50mM casein solution (pH 7.4) with the same volume as the magnetic bead solution is added into the magnetic bead solution, the mixture is ultrasonically mixed (40kHz, 6min), the mixture is placed on a shaking table and is subjected to shaking reaction at 37 ℃ and 100rpm for 24 min. After the reaction was completed, the reaction mixture was subjected to a strong magnetic separation using a 50mM casein solution (pH 7.4), and the supernatant was discarded and repeated twice.
4.1.5 preservation:
to the magnetic bead pellet obtained after the completion of blocking centrifugation, a stock solution (50mM Tris, 0.5 wt% BSA, pH7.8) was added so that the final magnetic bead concentration became 5mg/mL, and the mixture was sonicated (40kHz, 6min) and stored at 4 ℃.
4.1.6 vacuum drying:
and ultrasonically mixing the magnetic bead storage solution uniformly (40kHz, 6min), subpackaging the mixture into 5mL penicillin bottles according to 0.4 mL/bottle, sealing the vials by using gauze, and putting the vials into a vacuum freeze dryer to perform vacuum freeze drying for 10 hours at the temperature of between 10Pa and 50 ℃. The reagent can be stored at 4 deg.C for two years.
Example 5
When the immunomagnetic beads obtained in the embodiment 4 are used for separating red blood cells in blood plasma, 0.5mL of whole blood is added into the penicillin bottle in the embodiment 4, the mixture is shaken up and then placed on a magnetic frame or a magnet, and the upper layer of blood plasma is obtained after 2 min.
Example 6
Comparing the magnetic bead separation method with the standing method, and detecting the result difference by using a PCT kit.
Respectively taking 20 parts of fresh anticoagulated whole blood, uniformly dividing the anticoagulated whole blood into two groups, adding the immunomagnetic beads obtained in the embodiment 4 into one group, and then placing the groups on a magnetic frame to be marked as an immunomagnetic bead group; the other group is naturally placed with separated plasma and is marked as a natural placing group. After 2min, basically settling blood cells of the immunomagnetic bead group; while the natural setting group basically takes 20-30min to get plasma.
The two groups of obtained blood plasma are simultaneously detected by a CPR kit, and the detection results are compared with those of the two groups without obvious difference and are listed in Table 2.
TABLE 2
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Magnetic bead group (ng/ml) | 8.62 | 1.09 | 15.37 | 2.15 | 56.74 | 3.80 | 6.41 | 11.97 | 3.99 | 7.91 |
| Placing group (ng/ml) | 8.23 | 1.26 | 15.00 | 2.28 | 55.23 | 3.85 | 6.09 | 12.33 | 4.05 | 7.98 |
As can be seen from Table 2, the method for magnetically separating blood cells by using the immunomagnetic beads obtained by the invention is simple and effective, can rapidly separate blood cells to obtain blood plasma, has no residue, and has no influence on substances to be detected.
In the foregoing embodiments, the descriptions of the respective embodiments have respective emphasis, and for parts that are not described in detail in a certain embodiment, reference may be made to related descriptions of other embodiments.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (11)
1. A preparation method of immunomagnetic beads for separating red blood cells is characterized by comprising the following steps:
s1, activating magnetic beads by using an activating agent, wherein the weight ratio of the magnetic beads to the activating agent is 1: 0.1-5; the activation time is 5-30 min;
step S2, adding an anti-erythrocyte antibody into the activated product obtained in the step S1, and carrying out ultrasonic treatment;
and S3, adding a sealing agent into the product obtained in the step S2 for sealing, and performing strong magnetic separation to obtain the immunomagnetic beads for separating the red blood cells.
2. The method of claim 1, wherein the magnetic beads are microspheres coated with ferroferric oxide or ferric oxide, and the surfaces of the microspheres have carboxyl, amino or hydroxyl groups.
3. The method of claim 2, wherein the magnetic beads are polystyrene microspheres coated with ferroferric oxide.
4. The method of claim 1, wherein the magnetic beads have a particle size of 1-5 μm.
5. The method of claim 1, wherein the activator comprises at least one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, glutaraldehyde, periodate, and chloramine-T.
6. The method of claim 1, wherein in step S2, the mass ratio of the anti-erythrocyte antibodies to the magnetic beads is: 1: 10-1000.
7. A method for preparing an immunomagnetic bead for separating red blood cells according to claim 1, wherein in step S3, the blocking agent is at least one selected from the group consisting of 1-5 wt% bovine serum albumin solution, 0.1-5 wt% casein solution and 10-50mM glycine.
8. The method of claim 1, wherein the step S3 further comprises:
washing the obtained immunomagnetic beads for separating the red blood cells, adding the immunomagnetic beads into a preservation solution, uniformly dispersing, dividing into small parts according to the required amount, and carrying out vacuum drying, wherein the preservation solution comprises 50mM of tris (hydroxymethyl) aminomethane and 0.5 wt% of bovine serum albumin, and the pH value of the preservation solution is 7.8.
9. The method of claim 8, wherein the vacuum drying comprises: and carrying out vacuum freeze-drying treatment on the immunomagnetic beads under the pressure of 10-30Pa and the temperature of below-30 ℃.
10. An immunomagnetic bead for separating red blood cells, wherein the immunomagnetic bead is prepared by the preparation method of any one of claims 1 to 9.
11. Use of the immunomagnetic beads of claim 10 for separating red blood cells from plasma.
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| WO2022166230A1 (en) * | 2021-02-03 | 2022-08-11 | 杭州欣捷生物技术有限公司 | Composition and method for rapid separation of whole blood |
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